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Targeted detection of genetic alterations reveal the prognostic impact of H3K27M and MAPK pathway aberrations… Ryall, Scott; Krishnatry, Rahul; Arnoldo, Anthony; Buczkowicz, Pawel; Mistry, Matthew; Siddaway, Robert; Ling, Cino; Pajovic, Sanja; Yu, Man; Rubin, Joshua B; Hukin, Juliette; Steinbok, Paul; Bartels, Ute; Bouffet, Eric; Tabori, Uri; Hawkins, Cynthia Aug 31, 2016

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RESEARCH Open AccessTargeted detection of genetic alterationsreveal the prognostic impact of H3K27Mand MAPK pathway aberrations inpaediatric thalamic gliomaScott Ryall1,2 , Rahul Krishnatry1,3, Anthony Arnoldo4, Pawel Buczkowicz1,4, Matthew Mistry1, Robert Siddaway1,Cino Ling1, Sanja Pajovic1, Man Yu1, Joshua B. Rubin5, Juliette Hukin6, Paul Steinbok7, Ute Bartels3, Eric Bouffet3,Uri Tabori1,3,8 and Cynthia Hawkins1,2,4,9*AbstractPaediatric brain tumours arising in the thalamus present significant diagnostic and therapeutic challenges to physiciansdue to their sensitive midline location. As such, genetic analysis for biomarkers to aid in the diagnosis, prognosis andtreatment of these tumours is needed. Here, we identified 64 thalamic gliomas with clinical follow-up and characterizedtargeted genomic alterations using newly optimized droplet digital and NanoString-based assays. The median age atdiagnosis was 9.25 years (range, 0.63–17.55) and median survival was 6.43 (range, 0.01–27.63) years. Our cohortcontained 42 and 22 tumours reviewed as low and high grade gliomas, respectively. Five (12 %) low grade and 11(50 %) high grade gliomas were positive for the H3F3A/HIST1H3B K27M (H3K27M) mutation. Kaplan-Meier survivalanalysis revealed significantly worse overall survival for patients harbouring the H3K27M mutation versus H3F3A/HIST1H3B wild type (H3WT) samples (log-rank p < 0.0001) with a median survival of 1.02 vs. 9.12 years. Mitogen-activatedprotein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations or fusion events were detected in 44 %of patients, and was associated with long-term survival in the absence of H3K27M (log-rank p < 0.0001). Multivariateanalysis demonstrated H3K27M status and high grade histology to be the most significant independent predictors ofpoor overall survival with hazard ratios of 6.945 and 7.721 (p < 0.0001), respectively. In contrast, MAPK pathway activationis a predictor of favourable patient outcome, although not independent of other clinical factors. Importantly, we showthat low grade malignancies may harbour H3K27M mutations and that these tumours show a dismal survival comparedto low grade H3WT cases. Our data strongly supports the inclusion of targeted genetic testing in childhood thalamictumours to most accurately stratify patients into appropriate risk groups.Keywords: Thalamic glioma, Pediatric, H3K27M, MAPK, BRAF, PrognosticIntroductionBrain tumours are the largest group of solid tumoursand the leading cause of tumour-related death in chil-dren [4]. In particular, tumours arising in midlinestructures of the brain including the brainstem andthalamus present significant challenges for physiciansin regards to their therapeutic approach. At present,tumours arising in the brainstem, known as diffuseintrinsic pontine glioma (DIPG), have been extensivelyinvestigated and stratified into disease subtypes [6, 20].Whereas DIPG is heavily researched, thalamic tu-mours, accounting for approximately 5 % of all paedi-atric brain neoplasms, are largely unexplored [8–10,14, 27, 28]. Currently, the outcome of patients withthalamic tumours is predicted based on the histo-logical grade and the potential for surgical resectionof the tumour. High grade tumours classified asWorld Health Organization (WHO) grade III or IV* Correspondence: cynthia.hawkins@sickkids.ca1Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for SickChildren, Toronto, ON, Canada2Department of Laboratory Medicine and Pathobiology, University ofToronto, Toronto, ON, CanadaFull list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Ryall et al. Acta Neuropathologica Communications  (2016) 4:93 DOI 10.1186/s40478-016-0353-0are associated with worse clinical outcome as com-pared to grade I or II low grade tumours [23, 24].Due to their precarious midline location and the vitalfunctions of the thalamus and nearby structures, thesetumours, particularly the diffuse gliomas, often cannotbe completely resected, which is associated with aworse clinical outcome [2, 10, 22, 27]. Further, limitedresections also potentially lead to sampling related er-rors in histological grading due to small biopsy speci-mens [12, 26]. As such, there is a need to identifygenetic biomarkers to supplement histological gradingand extent of surgical resection to aid in the diagno-sis and management of thalamic tumour cases.Recent studies have identified several biomarkersthat aid in disease diagnosis and are important inpredicting patient outcome in childhood gliomas. Re-current mutations in histone H3 in which lysine 27is substituted for methionine (H3K27M) were firstdescribed in patients with paediatric high grade gli-oma, primarily DIPG [20, 31, 33]. When correlatedto clinical outcome, the presence of H3K27M inDIPG was associated with worse overall survival ascompared to those with wild type H3 (H3WT) sta-tus, regardless of the histology of the tumour [5, 20,21]. Subsequent studies have identified H3K27M mu-tations in other high grade midline tumours, includ-ing those in the thalamus, but have not lookeddirectly at its impact on patient survival [5, 31, 33].In addition to H3K27M, genetic aberrations affectingthe RAS-MAPK pathway including KIAA1549-BRAFand other BRAF, RAF and FGFR fusion events, aswell as BRAF and FGFR1 point mutations have beendescribed primarily in hemispheric low grade gliomasin adults and children [7, 18, 19, 30, 34]. KIAA1549-BRAF fusions have previously been shown to be as-sociated with better patient survival [3, 15] whileBRAFV600E has been linked to increased likelihoodof tumour progression and transformation in lowgrade glioma [17, 25]. FGFR1 aberrations includingN546K and fusion events with TACC were reportedin low grade paediatric astrocytomas [18, 34] andthe former shown to be a negative prognosticmarker in a small cohort of grade I pilocytic astrocy-tomas [3]. However, thalamic gliomas are oftenunder-represented in glioma cohorts because theirmidline location often means only a biopsy is per-formed and there is little tissue available to study.Thus a comprehensive study of genetic markers andtheir role relative to histologic and clinical risk fac-tors has not been performed for thalamic glioma. Toaddress this limitation, we assembled a cohort ofconcisely defined paediatric thalamic glioma. We in-vestigated the diagnostic and prognostic roles of de-fined genetic, clinical and histologic markers.Materials and methodsPatient cohortAfter institutional ethics board approval of the study, re-view of the pathology and oncology databases at the To-ronto Hospital for Sick Children (SickKids) identified101 patients diagnosed with thalamic glioma in the MRIera (1986 to 2014). As SickKids is the only referencecenter for children in a population of 5 million people,no selection bias is expected, and this qualifies as apopulation-based study. All cases were centrallyreviewed for pathological diagnosis and grading accord-ing to WHO criteria (CH) [23]. Where available, MRIswere reviewed to confirm thalamic tumour origin (RK).Twenty-three tumours originally identified as thalamicwere determined to not be central to or originating fromthe thalamus but rather, involved the thalamus and wereexcluded. Four cases were excluded due to bi-thalamicinvolvement. Ten tumours were excluded due to insuffi-cient material yielding a final cohort of 64 (Additionalfile 1: Table S1). We further assembled an independenttrans-Canadian cohort as previously described [32]which was used for validation purposes (Additional file 2:Table S2).DNA/RNA isolationDNA was extracted from 5 to 10 10 μm thick scrolls offormalin-fixed-paraffin-embedded (FFPE) tissue usingthe MasterPure Complete DNA and RNA PurificationKit (Epicentre, WI, USA) according to the manufac-turer’s instructions with a modified proteinase K diges-tion in which incubation time was increased from 24 to48 h. Total RNA was extracted from FFPE tissue withthe RNeasy FFPE extraction kit (QIAGEN, CA, USA)using the manufacturer’s guidelines. RNA/DNA qualitywas assessed based on 260/280 values obtained usingthe NanoDrop 2000 (Thermo Scientific, DE, USA) andsamples between 1.7–1.9 and 1.9–2.0 were considered tobe of sufficient quality for DNA and RNA, respectively.Samples were quantified using the Qubit FluorometerV2.0 (Thermo Scientific, DE, USA).ddPCR mutation detectionThe Bio-Rad (Hercules, CA, USA) QX200 ddPCR sys-tem was used to detect H3K27M (including H3F3A andHIST1H3B/C due to sequence similarity), H3F3A-G34V/R, BRAFV600E and FGFR1N546K mutations.Droplet digital PCR (ddPCR) workflow was completedas described [16]. Samples temporarily consisted of 1XddPCR Supermix for Probes (no dUTP) (Bio-Rad),900nM of specific forward and reverse primers, 250nMof specific mutant and wild type specific probe, and 10–50 ng of genomic DNA depending on the quality. Eachreaction was mixed with 70 μl of Droplet Generation Oil(Bio-Rad) and partitioned into a minimum of 10,000Ryall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 2 of 10droplets, transferred to a 96-well plate and sealed priorto PCR amplification. PCR amplification was completedin a C1000 thermo cycler (Bio-Rad) with the followingcycling conditions: 1 x (95 °C for 10 min), 39 x (95 °Cfor 30 s, 55 °C for 60 s, with 2 °C s-1 ramp rate), and 1 x(98 °C for 10 min). Following amplification, fluorescentintensity was measured with the QX200 Droplet Reader(Bio-Rad) and data analysis performed with the Quanta-Soft droplet reader software (Bio-Rad). All samples wererun in duplicate to ensure validity. Samples were consid-ered positive if a minimum of 10 mutant droplet and1 % mutant allele frequency were detected in both dupli-cate runs. This threshold was arbitrarily set and we re-port no false positive detection ever approaching thisdefined threshold.The ddPCR assay was fully optimized for clinical im-plementation. DNA loading concentration, PCR settings,and reaction conditions were corrected to ensure suffi-cient probe separation for confident mutation identifica-tion (Additional file 3: Figure S1, Additional file 4:Figure S2, and Additional file 5: Figure S3). A cohort ofclinical DIPG and high grade astrocytoma samples previ-ously evaluated with whole exome sequencing (WES)(Applied Biosystems SOLiD 5500xl) were used to com-pare methods [20]. ddPCR showed 100 % concordancewith WES calls from both fresh frozen and FFPE sam-ples (Additional file 6: Table S3 and Additional file 7:Table S4). Importantly, it must be noted that due to se-quence similarity, the H3K27M droplet digital probe isunable to distinguish between H3F3A and HIST1H3B/C, and hence will detect a K27M mutation in any of theaforementioned genes. Similar optimization procedureswere completed for H3G34V/R, BRAFV600E andFGFR1N546K (data not shown). Assay sensitivity wasdetermined by serially diluting an H3K27M-positivesample with normal DNA from 50 to 0.01 % MAF(Additional file 8: Figure S4). The ddPCR assay was ableto accurately detect the mutation at allele frequencies aslow as 1 % in FFPE preserved samples in accordance withthe minimum detection limits described above.NanoString fusion detectionProbes targeting the 33 most commonly reported fusionsin paediatric glioma (Additional file 9: Table S5) weredesigned in collaboration with NanoString (WA, USA).Five hundred nanograms of total RNA was added to thenCounter Elements TagSet in hybridization buffer andincubated at 67 °C for 20 h. The sample was processedon the nCounter Preparation Station and the cartridgescanned at 555 fields of view on the nCounter DigitalAnalyzer. Raw counts were subjected to a technicalnormalization using counts obtained for positive controlprobe sets included in each run. The statistical outlierdetection method was used to detect the presence of anexpressed fusion. Data is viewed using a box plot andthe presence of an extreme outlier (3xIQR) indicate fu-sion expression.StatisticsStatistical analysis was performed using SPSS v23 (IBMCorporation) or GraphPad Prism 5 (La Jolla, CA, USA).Overall survival was determined using the Kaplan–Meiermethod and univariate assessments of Kaplan–Meierplots were tested using log rank. p values <0.05 wereconsidered to be statistically significant. MultivariateCox proportional hazard models and significance basedon the Wald test (α = 0.05) were performed for multi-variate analysis.ResultsClinical characteristics of patients with thalamic gliomaSixty-four patients treated at the Hospital for SickChildren from 1986 to 2014 were identified as thalamictumour patients. Forty-two thalamic tumours were his-tologically diagnosed as low grade glioma (62 % grade I,5 % grade II, and 33 % low grade glioma, NOS) whereasthe remaining 22 were diagnosed as high grade glioma(41 % grade III, 50 % grade IV, and 9 % high grade,NOS). Two (5 %) low grade gliomas later transformed tohigh grade malignancies. Median age of diagnosis forthalamic glioma patients was 9.25 years (range, 0.63–17.55 years). Forty-one (64 %) patients received surgicalresection (5 partial, 25 subtotal and 11 gross total resec-tion) while 23 (36 %) were biopsied only. Thirty-five(55 %) and 37 (58 %) patients were treated with chemo-therapy and/or radiation, respectively. Thirty-five (55 %)patients are alive (median follow-up, 12.2 years) while 29(45 %) patients succumbed to their disease. A summaryof the clinical characteristics are shown in Table 1. Clin-ical characteristics of the Canadian cohort reflect thosedescribed above and are available in Additional file 10:Table S6.Landscape of point mutations and fusion events inthalamic tumoursddPCR and NanoString assays were used to identify tar-geted mutations and fusions of interest based on theirprevious association with paediatric glioma (Fig. 1). Themost recurrent hotspot mutation was H3K27M, identi-fied in 16 (25 %) thalamic tumours tested. No H3G34R/V mutations were observed as expected. BRAFV600Emutations were present in 10 (16 %) cases, with 2 co-occurring with H3K27M mutations. KIAA1549-BRAFfusion events including those involving exons 16;09,16;11 and 15;09 in order of prevalence, were detected in14 (39 %) of the 36 samples from which sufficient qualityRNA was obtained. These were mutually exclusive withH3K27M, BRAFV600E and FGFR fusions/mutations.Ryall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 3 of 10FGFR1N546K mutations were seen in 4 (6 %) tumoursamples. No FGFR1-TACC1, FGFR3-TACC3, otherBRAF or RAF fusions or MYBL1 alterations were de-tected in this cohort. Within the Canadian cohort,H3K27M was identified in 5 (31 % of patients),BRAFV600E in 3 (19 %) and KIAA1549-BRAF fusionevents in 3 (20 %) of 15 patients tested. NoFGFR1N546K or FGFR1-TACC1, FGFR3-TACC3, otherBRAF or RAF fusions or MYBL1 alterations were de-tected within this cohort. Genetic aberration frequenciesbased on histological grade can be seen in Additionalfile 11: Table S7.High grade histology and H3K27M are markers of poorprognosis in thalamic gliomaTumours diagnosed as high grade glioma (grades III andIV) showed significantly worse overall survival whencompared to those defined as low grade (grade I and II)in both the SickKids and Canadian cohorts (Additionalfile 12: Figure S5). This trend was conserved with the re-moval of PA and GG from the low grade histology co-hort (log-rank p = 0.0027). 5+ year overall survival forpatients diagnosed with a high grade glioma was 9.1 %(2/22) as compared to 76.2 % (32/42) in tumours withlow grade histology. 9.1 % and 78.6 % of high and lowgrade patients respectively were alive at the time thestudy was completed. Likewise, the presence ofH3K27M was significantly related to worse patient out-come (Fig. 2a, log-rank p < 0.0001). Patients harbouringthe H3K27M mutation had a 5+ year overall survival of6.3 % (1/16) as compared to 68.8 % (33/48) for H3WTpatients (Table 2). Similar results were observed in theCanadian cohort (Fig. 2b, log-rank p = 0.0002). Of the 16H3K27M positive cases, 5 and 11 were low and highgrade glioma, respectively. All 16 of these patients suc-cumbed to their disease. No patient with a morphologic-ally classic pilocytic astrocytoma harboured an H3K27Mmutation, suggestive of a minimum grade II histology inNOS cases.H3K27M is present in low grade thalamic glioma andconfers a negative prognosisWhen separated based on histological grade, bothH3K27M and H3WT high grade glioma cases showed poorprognosis (Fig. 2c), with tumours harbouring H3K27M hav-ing slightly worse outcome (log-rank p = 0.0109). Longterm survival (5+ years) in high grade cases was exclusivelyseen in H3WT patients. Strikingly, H3K27M positive lowgrade cases had significantly worse outcome than H3WTlow grade tumours (log-rank p < 0.0001), with all H3K27Mpatients succumbing to their disease (Fig. 2d). Upon re-moval of PA and GG from the low grade cohort, the pres-ence of H3K27M remained a predictor of a worse patientoutcome (log-rank p =0.0195). Interestingly, whencompared to high grade H3K27M glioma patients, pa-tient survival was significantly longer for patients withlow grade glioma histology (median survival 1.44[range, 0.52–13.66] and 0.76 [range, 0.12–1.52] yearsfor low grade versus high grade respectively, log-rankp = 0.0361) suggesting that under-grading due to sam-pling error does not account for the finding ofTable 1 Clinical characteristics of paediatric thalamic gliomaCharacteristic Number of patientsSexMale 34Female 30OutcomeAlive 35Dead 29GradeLow Grade 42High Grade 22HistologyPilocytic 23Diffuse 2Anaplastic 9Glioblastoma 11Ganglioglioma 3Low Grade, NOS 14High Grade, NOS 2Extent of SurgeryGTR 11STR 25Partial Resection 5Biopsy 12Unknown 11RadiationTreated 37Not Treated 22Unknown 5ChemotherapyTreated 35Not Treated 26Unknown 3Age at DiagnosisMedian 9.25 yearsMean 8.77 ± 3.86 yearsOverall SurvivalMedian 6.43 yearsMean 8.78 ± 8.68 yearsRyall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 4 of 10H3K27M in low grade glioma. MRI review of availablecases showed no obvious differences within the low gradeH3K27M cases to suggest that a high grade tumour waspresent. Importantly, of patients with low grade thalamicgliomas and H3WT genotype, 89 % were alive at the com-pletion of this study (median follow-up 12.2 years). Fourlow grade H3WT cases succumbed to their disease, all ofwhich were WT for all aberrations tested in this study.Taken together, the presence of low grade histology andH3WT status resulted in the most favourable patient out-come, while high grade histology and H3K27M resulted inthe worst outcome (Fig. 2e). Low grade histology in thepresence of H3K27M as well as high grade H3WT casesact intermediately. Similar clinical outcome in H3K27Mand H3WT cases were observed within the Canadian co-hort (Additional file 13: Table S8).MAPK pathway activation is a marker of good prognosisin thalamic gliomaMAPK pathway activation via BRAF and FGFR1 hotspotmutations or fusion events was detected in 28 (44 %) ofchildhood thalamic gliomas. BRAFV600E was detectedin 2 and 8 high and low grade gliomas, respectively. Ofthe 2 high grade patients with BRAFV600E, one patientalso harboured H3K27M and had poor survival(1.43 years) in line with the H3K27M phenotype. Anec-dotally, the patient with H3WT high grade glioma andBRAFV600E is still alive and has a longer survival com-pared to most other high grade tumours (3.75 yearsfollow-up). In low grade gliomas, the presence ofBRAFV600E was associated with excellent overall sur-vival (10.27 [range, 1.27–24.95] year median survival);the only patient (1) deceased by the study endpoint wasMale Female High Grade Low Grade Sex  Histology Genetics  Wild Type   Mutant   Insufficient Quality/Material   Cohort  SickKidsCanadian   SexHistologyCohortH3K27MH3G34RH3G34VBRAFV600EFGFR1N546KKIAA1549-BRAFFGFR1-TACCFig. 1 Genetic, molecular and clinical characteristics of paediatric thalamic gliomap<0.0001A Bp=0.0002Cp<0.0001Dp=0.0109Discovery Cohort Validation CohortHigh Grade Tumours Low Grade Tumours0 5 1 0 1 505 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalH 3 K 2 7 MH 3 W T0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalH 3 K 2 7 MH 3 W Tp<0.001E0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalL o w G r a d e + H 3 W TL o w G r a d e + H 3 K 2 7 MH ig h G r a d e + H 3 W TH ig h G r a d e + H 3 K 2 7 M0 5 1 0 1 5 2 0 2 505 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalH 3 K 2 7 MH 3 W T0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalH 3 K 2 7 MH 3 W TFig. 2 H3K27M is a negative prognostic marker in paediatric thalamic glioma. SickKids cohort (a) and )(b) Canadian cohort were tested forassociation between H3K27M-status and survival. Sample stratification based on histological grade showed H3K27M a significant prognosticmarker in high grade (c) and low grade (d). Histology and H3K27M status combined (e) revealed clinical disease stratificationRyall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 5 of 10also H3K27M-positive as compared to 100 % 5-year sur-vival of all other cases (Fig. 3a). BRAF fusion events wereexclusively seen in low grade tumours, were never asso-ciated with H3K27M and were associated with excellentlong-term survival (5+ year survival of 78.6 % [11/14])(Fig. 3b). All patients harbouring a BRAF fusion eventwere alive upon completion of this study (mean follow-up 13.02 years). FGFR1 mutations were found in 6 % ofthalamic gliomas (5 % and 9 % of low and high grade tu-mours respectively). FGFR1 mutations were observed toco-occur with H3K27M in two cases and as withBRAFV600E, the H3K27M phenotype of poor survivalwas observed. The FGFR1 positive, H3K27M negativepatients behaved in accordance to their histologicalgrade, with the grade IV case succumbing to the diseasein 1.71 years while the grade I surviving 25.46 years atthe time of last follow-up. FGFR1 mutations were mutu-ally exclusive with BRAFV600E and BRAF fusion eventsin this series. Overall, the presence of MAPK pathwayactivation in the absence of H3K27M was related to ro-bust patient survival (91 % 5-year survival), whereas inthe presence of H3K27M the prognosis remained pooracross all histological grades (Fig. 3c).H3K27M status, extent of resection and histological gradeare independent predictors of patient survival in thalamicgliomaTo determine their relative predictive values, we per-formed univariate analysis of H3K27M, MAPK path-way activation, extent of surgical resection, adjuvanttherapy and histological grade. On univariate analysishistological grade (high grade versus low grade, HR9.905 [range 4.378–22.408], p < 0.0001), extent of re-section (resection versus biopsy, HR 0.399 [range0.191–0.831], p = 0.014) and H3K27M status (K27Mversus WT, HR 9.403 [range 4.253–20.788], p < 0.001)and MAPK pathway activation (Activated versus WT,HR 0.190 [range 0.072–0.500], p = 0.001 are predictivemarkers of overall survival (Table 3). On multivariateanalysis, only H3K27M status (HR 6.945, p < 0.0001),histologic grade (HR 7.721, p < 0.0001) and extent ofsurgical resection (HR 0.325, p = 0.025) were signifi-cant independent predictors of patient survival.DiscussionThalamic tumours present a significant challenge for cli-nicians in terms of accurate diagnosis and an appropri-ate therapeutic approach. This study investigated theprognostic potential of molecular alterations and clinicalfactors in a concisely defined thalamic glioma cohort.As expected, our results show the presence ofH3K27M mutations in 50 % of high grade paediatricthalamic tumours [5, 20, 31, 33]. Interestingly, 12 % ofpatients diagnosed with low grade thalamic tumours alsotested positive for H3K27M despite previous reportssuggesting it to be exclusive to high grade cases whennot located in the brainstem [31, 33]. Of note, two offive low grade cases positive for H3K27M laterTable 2 Clinical characteristics of H3K27M and H3WT paediatricthalamic gliomaCharacteristic Number of patientsH3WT H3K27M48 16SexMale 28 6Female 20 10OutcomeAlive 35 0Dead 13 16HistologyLow Grade 37 5High Grade 11 11GradePilocytic 23 0Diffuse 0 2Anaplastic 6 3Glioblastoma 5 6Ganglioglioma 3 0Low Grade, NOS 11 3High Grade, NOS 0 2Extent of SurgeryGTR 11 0STR 19 6Partial Resection 3 2Biopsy 7 5Unknown 8 3RadiationTreated 29 8Not Treated 16 6Unknown 3 2ChemotherapyTreated 29 6Not Treated 17 9Unknown 2 1Age at DiagnosisMedian 9.02 years 10.50 yearMean 8.24 ± 3.93 years 10.35 ± 3.23 yearsOverall SurvivalMedian 9.12 years 1.02 yearsMean 11.10 ± 8.69 years 1.81 ± 3.25 yearsRyall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 6 of 10transformed to high grade malignancies at the time ofsecond surgery, a rare occurrence in paediatric lowgrade glioma and consistent with the diagnosis of sec-ondary HGG [25]. While it is possible that theseH3K27M positive thalamic gliomas were under-gradedhistologically based on sampling bias, the significant sur-vival difference observed between low grade and highgrade H3K27M tumours supports the idea that these tu-mours were indeed distinct from their high grade coun-terparts. Further, there is a lack of any distinguishingMRI characteristics to suggest under-grading in thesecases. Importantly, under the new World HealthOrganization classifications published recently, thesetumours would be classified as diffuse midline glioma,H3-K27M mutant, further supporting their uniqueidentity as compared to non-H3K27M low grade tu-mours [24].Patients harbouring H3K27M showed significantlyworse overall survival when compared to H3WT cases.Once separated based on histological grade, both lowand high grade tumours maintained a significantly worsesurvival in the presence of H3K27M. Of note, high gradethalamic tumours, regardless of H3K27M status, yieldeddismal survival with those harbouring the mutation suc-cumbing to their disease in a slightly faster timeframe.This result coincides with our previous work in DIPGp<0.0001AP=0.2478Bp<0.0001C0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalB R A F V 6 0 0 EB R A F V 6 0 0 E + H 3 K 2 7 MW ild T y p eH 3 K 2 7 M0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalW i ld T y p eB R A F F u s io n0 1 0 2 0 3 005 01 0 0O v e r a l l S u r v iv a l ( y e a r s )PercentSurvivalM A P K A c t iv a t io nM A P K A c t iv a t io n + H 3 K 2 7 MH 3 K 2 7 MW ild T y p eFig. 3 MAPK pathway activation is a positive predictive marker. BRAFV600E in the absence of H3K27M in low grade tumours. a shows a survivaladvantage. BRAF fusion events in low grade tumours. b shows robust survival. MAPK activation combined. c shows reveals a positive prognosisthroughout low and high grade tumoursTable 3 Univariate and multivariate Cox analysis of genetic and clinical determinants of paediatric thalamic gliomaVariable Univariate (95 % CI) p value Multivariate (95 % CI) p valueHistology (HG vs. LG) 9.905 [4.378–22.408] <0.0001 7.721 [2.437–24.461] <0.0001Surgery (resection vs. biopsy) 0.399 [0.191–0.831] 0.014 0.325 [0.122–0.869] 0.025Chemo. (yes vs. no) 0.969 [0.441–2.127] 0.969 0.549 [0.213–1.416] 0.215Rad. (yes vs. no) 1.048 [0.459–2.396] 0.911 0.898 [0.343–2.349] 0.827H3K27M (mut. vs. WT) 9.403 [4.253–20.788] <0.0001 6.945 [2.190–22.020] <0.0001MAPK Activation (mut. vs. WT) 0.190 [0.072–0.500] 0.001 0.385 [0.134–1.102] 0.075LG low grade, HG high gradeRyall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 7 of 10[5, 20], where we found H3K27M to be a negative prog-nostic marker in DIPG, albeit independent of tumourhistology. Previous research investigating the impact ofH3K27M on high grade adult midline tumours found acorrelation between H3K27M and poor survival in thebrainstem, but not the thalamus [1, 13]. Similar to thisstudy, in our cohort high grade histology was associatedwith a poor outcome in both H3K27M and H3WT pa-tients. However, in our cohort, several longer term survi-vors with H3WT high grade gliomas were presentmaking the overall survival slightly better for H3WTpatients. Work investigating paediatric glioblastoma(Grade IV) identified H3K27M positive cases asshowing poor survival in midline cases includingthose in the brainstem and thalamic regions, consist-ent with our findings [21].The presence and effect of H3K27M mutations in lowgrade malignancies on patient outcome has not previ-ously been shown in malignancies outside the brainstem.In this study, patients with low grade thalamic gliomashad good overall survival with 79 % of patients aliveupon the completion of this study (mean follow-up14.03 years), consistent with previous studies [3, 11, 15,29, 30]. However, all 5 patients whose low grade gliomaswere positive for H3K27M succumbed to their disease,with increased latency compared to high grade H3K27Mcases. These findings substantiate the knowledge thatH3K27M mutations do exist in low grade tumours andthat H3K27M status can supplement histological gradingin determining the clinical progression of the tumour.Importantly, the poor survival of patients with H3K27Mlow grade glioma suggests that they should be treated ashigh grade glioma; receiving more aggressive adjuvanttherapies than would be typically given to low grade gli-oma patients.MAPK pathway activation was detected in 44 % of pa-tients. BRAFV600E was detected in 10 (16 %) of thethalamic tumour samples, a slightly lower percentagethan previously reported [29, 30]. However, in these re-ports, the thalamus was grouped with all diencephalon-based structures, potentially altering the true prevalence.Furthermore, this study identified the presence ofBRAFV600E mutations in 3 grade I malignancies for thefirst time, in contrast with previous reports [11]. In theabsence of H3K27M, BRAFV600E cases were associatedwith a good clinical outcome. Previously we reportedthat low grade tumours having BRAFV600E were at ahigher risk for developing secondary high grade glioma[25]. However, only 3 patients within this initial studywere identified as thalamic, of which one co-occurredwith H3K27M and another with CDKN2A deletion, po-tentially predisposing to tumour transformation. Fur-thermore, although the results here suggest robustsurvival in our BRAFV600E cohort, our median follow-up was 10.27 years and it is possible that with longer la-tency these patients may develop high grade glioma.Therefore, patients harboring low grade BRAFV600E tu-mours should be monitored closely post-treatment fordisease progression. In the presence of H3K27M,BRAFV600E patients had an outcome similar toBRAFWT, H3K27M-positive cases, suggesting thatH3K27M is the dominant prognostic indicator.KIAA1549-BRAF fusion events were exclusive to lowgrade thalamic glioma and were found in 39 % of testedtumours. This percentage is similar to previous reportsin midline paediatric low grade glioma [11, 15]. How-ever, it should be noted that these studies included non-thalamic locations, including the cerebellum, hypothal-amus, optic pathway and brainstem. Patients with aKIAA1549-BRAF fusion showed robust survival, with all14 patients alive at the time this study was completed(median follow-up: 11.55 years). FGFR1N546K was de-tected in 4 patients (6 %) and was seen in both low andhigh grade cases. FGFR1N546K mutations have beenpreviously linked to decreased patient survival [3]. Thesmall number of patients harbouring this mutation inthis cohort means conclusions related to its prognosticsignificance cannot be drawn. However, of the 4 FGFR1mutated patients, the 2 also harbouring H3K27Mshowed poor survival in accordance with the H3K27Mphenotype. Alternately, FGFR1 mutated patients withoutH3K27M behaved in accordance with their histologicalgrade, suggesting that histology and H3K27M status arethe main phenotypic contributors in these cases. We didnot identify FGFR1-TACC1 or FGFR3-TACC3 fusions inour cohort as described in [34].The presence of MAPK pathway activation was foundin 28 patients within our cohort, 89 % of which werelow grade glioma. Importantly, of the 3 high grade caseswith MAPK activation, 2 were also H3K27M and be-haved in accordance with the H3K27M phenotype. Theremaining high grade, MAPK activated sample shows aprolonged survival in comparison to other high gradetumours (3.75 years) and was alive upon the completionof this study. In low grade tumours, the presence ofMAPK activation, regardless of fusion or hotspot muta-tion event was related to prolonged survival. Taken to-gether, H3K27M and MAPK pathway activationeffectively stratifies thalamic tumours into survivalgroups. MAPK pathway activation in the absence ofH3K27M confers long-term survival across the entirecohort irrespective of tumour histology. Tumours wildtype for the genetic targets tested here behave in closeaccordance with their histological grade. Further testingis required to identify additional genetic marks capableof further stratifying this group. Importantly, it must berecognized that a limitation of this study in respect toMAPK activation is the inclusion of pilocyticRyall et al. Acta Neuropathologica Communications  (2016) 4:93 Page 8 of 10astrocytoma and ganglioglioma within the low gradehistology category. This saturation of circumscribed andnon-invasive lesions positive for MAPK activation maypartially explain the robust survival seen. However, inthe case of thalamic tumours, the finding appear consist-ent across histological grades and as such, remain an im-portant clinical predictor of patient outcome. Lastly,tumours harbouring H3K27M, regardless of histology orMAPK activation show dismal survival. In this respect,H3K27M is one of the most critical factors in predictingpatient outcome in thalamic glioma cases and must beconsidered equally important as tumour histology in pri-mary prognostic categorization.ConclusionsIn summary, this study supports the inclusion of tar-geted H3K27M and MAPK activation testing via clinic-ally approved methodology in all paediatric midlinetumours regardless of histological grade. This helps pro-vide an accurate prediction of patient outcome and ap-propriate therapeutic options. Further, we provide fullyoptimized and cost-effective strategies to targeting theseaberrations in a clinical setting.Additional filesAdditional file 1: Table S1. SickKids cohort details. (XLSX 17 kb)Additional file 2: Table S2. Canadian cohort details. (XLSX 11 kb)Additional file 3: Figure S1. Droplet digital PCR minimumconcentration detection. (PPTX 55 kb)Additional file 4: Figure S2. Droplet digital PCR annealing temperaturecomparison. (PPTX 173 kb)Additional file 5: Figure S3. Droplet digital PCR pre-PCR digestionversus no digestion comparison. (PPTX 102 kb)Additional file 6: Table S3. Droplet digital PCR H3K27M detectionvalidation as compared to WES and Sanger Sequencing. (DOCX 12 kb)Additional file 7: Table S4. Droplet digital PCR H3K27M detectionvalidation raw droplet counts. (XLSX 12 kb)Additional file 8: Figure S4. Droplet digital PCR minimum mutantallele frequency detection. (PPTX 38 kb)Additional file 9: Table S5. NanoString low grade glioma fusion andduplication detection panel. (XLSX 10 kb)Additional file 10: Table S6. Clinical characteristics of paediatricthalamic glioma Canadian cohort. (DOCX 12 kb)Additional file 11: Table S7. Histology Kaplan-Meier survival analysiswithin the A) SickKids cohort and B) Canadian cohort. (DOCX 12 kb)Additional file 12: Figure S5. Clinical characteristics of H3K27M andH3WT paediatric thalamic glioma Canadian cohort. (PPTX 84 kb)Additional file 13: Table S8. Clinical characteristics of H3K27M andH3WT paediatric thalamic glioma in the Canadian cohort. (DOCX 12 kb)AcknowledgementsThis research was supported by an operating grant from the CanadianCancer Society Research Institute [MOP 702296]. Scott Ryall is a recipient ofthe Canadian Institutes of Health Research Frederick Banting and CharlesBest Canada Graduate Scholarships Award. Rahul Krishnatry is a recipient ofthe Society of Neurooncology International Research Development Award.We would like to thank the patients and their families for donatingbiological material to allow this study to be conducted.Authors’ contributionsSR, UT and CH designed the study. SR, PB, SP, UT, and CH contributed towriting the manuscript. SR, RK, AA, UB, EB, UT, CH participated in cohortcompilation and sample preparation. SR, AA, MM, and CL completed thegenetic studies. SR, RK, UB, EB, UT, and CH provided the detailed clinicalfollow-up of the patients. JBR, JH, and PS provided biological material for thevalidation cohort. SR, AA, PB, MM, RS, MYand CH completed the statisticalanalysis. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationThis work has not been previously published.Author details1Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for SickChildren, Toronto, ON, Canada. 2Department of Laboratory Medicine andPathobiology, University of Toronto, Toronto, ON, Canada. 3Division ofHaematology/Oncology, Hospital for Sick Children, Toronto, ON, Canada.4Division of Pathology, Hospital for Sick Children, Toronto, ON, Canada.5Department of Paediatrics, Washington University School of Medicine in St.Louis, St. Louis, MO, USA. 6Division of Neurology and Oncology, Departmentof Paediatrics, University of British Columbia & British Columbia Children’sHospital, Vancouver, BC, Canada. 7Division of Paediatric Neurosurgery,Department of Surgery, University of British Columbia & British ColumbiaChildren’s Hospital, Vancouver, BC, Canada. 8Institute of Medical Science,University of Toronto, Toronto, ON, Canada. 9The Hospital for Sick Children,555 University Avenue, Toronto, ON M5G 1X8, Canada.Received: 25 July 2016 Accepted: 26 July 2016References1. 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